Autologous Neutralizing Antibodies to the Transmitted/Founder Viruses Emerge Late after Simian Immunodeficiency Virus SIVmac251 Infection of Rhesus Monkeys

While the simian immunodeficiency virus (SIV)-infected rhesus monkey is an important animal model for human immunodeficiency virus type 1 (HIV-1) infection of humans, much remains to be learned about the evolution of the humoral immune response in this model. In HIV-1 infection, autologous neutralizing antibodies emerge 2 to 3 months after infection. However, the ontogeny of the SIV-specific neutralizing antibody response in mucosally infected animals has not been defined. We characterized the kinetics of the autologous neutralizing antibody response to the transmitted/founder SIVmac251 using a pseudovirion-based TZM-bl cell assay and monitored env sequence evolution using single-genome amplification in four rhesus animals that were infected via intrarectal inoculations. We show that the SIVmac251 founder viruses induced neutralizing antibodies at 5 to 8 months after infection. Despite their slow emergence and low titers, these neutralizing antibodies selected for escape mutants that harbored substitutions and deletions in variable region 1 (V1), V2, and V4 of Env. The neutralizing antibody response was initially focused on V4 at 5 to 8 months after infection and then targeted V1/V2 and V4 by 16 months. These findings reveal a striking delay in the development of neutralizing antibodies in SIVmac-infected animals, thus raising questions concerning the suitability of SIVmac251 as a challenge strain to screen AIDS vaccines that elicit neutralizing antibodies as a means to prevent virus acquisition. They also illustrate the capacity of the SIVmac quasispecies to modify antigenic determinants in response to very modest titers of neutralizing antibodies.While neutralizing antibodies (Nabs) mediate protection in humans against a diversity of viral pathogens (38, 53, 72), it is unclear how they impact human immunodeficiency virus type 1 (HIV-1) infection. One reason that the contribution of neutralizing antibodies to the control of HIV-1 remains uncertain is that HIV-specific neutralizing antibodies develop relatively late in natural infection. High titers of HIV-specific autologous neutralizing antibodies usually emerge as late as 2 to 3 months after infection and continue to evolve throughout the first years of infection (18, 25, 57, 66, 74). Such neutralizing antibodies have been shown to influence HIV-1 evolution within a host and to be responsible for viral escape mutations (47, 48, 58, 59). A better understanding of why there is a prolonged time associated with the maturation of the neutralizing antibody response in HIV-1 infection and whether conserved viral epitopes exist that could mediate antibody protection is important for the development of effective HIV-1 vaccine strategies.The simian immunodeficiency virus (SIV)/rhesus macaque model of AIDS provides an important system to study AIDS immunopathogenesis and to evaluate HIV-1 vaccine strategies. SIVmac251, an uncloned, pathogenic, CCR5-tropic virus isolate comprised of a swarm of quasispecies that are closely related (33), and SIVmac239, an infectious molecular clone derived from SIVmac251, are the two most commonly used rhesus monkey SIV challenge viruses utilized in AIDS vaccine research in the nonhuman primate (NHP) model. SIVmac239 has been shown to be relatively resistant to antibody-mediated neutralization by both autologous antibodies and a wide range of monoclonal antibodies (29, 30). The env sequence evolution in SIVmac239-infected rhesus monkeys and SIVMne-CL8-infected pigtailed macaques has been well described (8, 50, 51). Some of these changes in Env have been shown to result in viral escape from neutralizing antibodies (7, 10, 34, 60). In particular, a recent study by Sato et al. characterized SIVmac239 env sequence changes that were associated with viral escape in a rhesus monkey with an unusually high titer of neutralizing antibodies after intravenous infection (67). However, the antibody-mediated neutralization of SIVmac251 has not been tested rigorously using standardized assays that are currently being used to measure neutralization of HIV-1, thereby precluding a direct comparison of the neutralization sensitivities of HIV-1 and SIV. Furthermore, it is also unclear whether more typical titers of neutralizing antibodies against SIV239/251 exert selection pressure on the viral population in animals that acquire infection mucosally.The aims of this study were to elucidate the kinetics of the neutralizing antibody response against the transmitted viruses and the sequence evolution of env in association with humoral immunity in mucosally infected rhesus macaques. We hypothesized that a low titer of SIVmac Env-specific neutralizing antibodies exerts potent selection pressure on the viral quasispecies. To test this hypothesis, we utilized a pseudovirion-based TZM-bl reporter gene neutralization assay and single genome amplification (SGA) in order to characterize the humoral immune pressures driving viral sequence evolution in four rhesus monkeys that were infected with SIVmac251 via intrarectal inoculations.     

Mucosal Trafficking of Vector-Specific CD4+ T Lymphocytes following Vaccination of Rhesus Monkeys with Adenovirus Serotype 5

Post hoc analysis of the phase 2b Step study evaluating a recombinant adenovirus serotype 5 (rAd5)-based HIV-1 vaccine candidate suggested a potential increased risk of HIV-1 acquisition in subjects who were baseline Ad5 seropositive and uncircumcised. These concerns had a profound impact on the HIV-1 vaccine development field, although the mechanism underlying this observation remains unknown. It has been hypothesized that rAd5 vaccination of baseline Ad5-seropositive individuals may have resulted in anamnestic, vector-specific CD4⁺ T lymphocytes that could have trafficked to mucosal sites and served as increased targets for HIV-1 infection. Here we show that Ad5-specific CD4⁺ T lymphocyte responses at mucosal sites following rAd5-Gag/Pol/Nef vaccination were comparable in rhesus monkeys with and without baseline Ad5 immunity. Moreover, the total cellular inflammatory infiltrates and the CD3⁺, CD4⁺, HLA-DR⁺, Ki67⁺, and langerin⁺ cellular subpopulations in colorectal and foreskin mucosa were similar in both groups. Thus, no greater trafficking of Ad5-specific CD4⁺ T lymphocytes to mucosal target sites was observed following rAd5 vaccination of rhesus monkeys with baseline Ad5 immunity. These findings from this nonhuman primate model provide evidence against the hypothesis that recruitment of vector-specific target cells to mucosal sites led to increased HIV-1 acquisition in Ad5-seropositive, uncircumcised vaccinees in the Step study.The Step study revealed a potential increased risk of HIV-1 acquisition among adenovirus serotype 5 (Ad5)-seropositive, uncircumcised subjects who received the Merck recombinant Ad5 (rAd5)-Gag/Pol/Nef vaccine candidate (2, 6). It has been hypothesized that rAd5 vaccination of Ad5-seropositive individuals may have resulted in robust expansion and activation of vector-specific CD4⁺ T lymphocytes that could have trafficked to mucosal sites and served as increased targets for HIV-1 infection. Our laboratory and others have recently demonstrated that total and vector-specific CD4⁺ T lymphocytes in peripheral blood in Ad5-seropositive volunteers were comparable to or lower than the levels in Ad5-seronegative volunteers following rAd5-Gag vaccination in the Merck phase 1 studies (4, 8). However, mucosal biopsy specimens were not obtained in these clinical trials, and thus the extent of inflammatory infiltrates and vector-specific CD4⁺ T lymphocytes in colorectal and foreskin mucosa could not be evaluated in these prior studies.It has also recently been reported that vector-specific CD4⁺ T lymphocytes may upregulate mucosal homing integrin expression following exposure to Ad5 in short-term in vitro cultures (1). These findings highlight the importance of directly investigating the extent and nature of vector-specific CD4⁺ T lymphocytes at mucosal sites following rAd5 vaccination. Given the lack of mucosal biopsy samples from human subjects in the Step study, we developed a nonhuman primate model of preexisting adenovirus immunity to evaluate the extent and nature of inflammatory cell populations at mucosal sites following rAd5 vaccination.     

Micronuclei in Kif18a mutant mice form stable micronuclear envelopes and do not promote tumorigenesis

Micronuclei, whole or fragmented chromosomes spatially separated from the main nucleus, are associated with genomic instability and have been identified as drivers of tumorigenesis. Paradoxically, Kif18a mutant mice produce micronuclei due to asynchronous segregation of unaligned chromosomes in vivo but do not develop spontaneous tumors. We report here that micronuclei in Kif18a mutant mice form stable nuclear envelopes. Challenging Kif18a mutant mice via deletion of the Trp53 gene led to formation of thymic lymphoma with elevated levels of micronuclei. However, loss of Kif18a had modest or no effect on survival of Trp53 homozygotes and heterozygotes, respectively. Micronuclei in cultured KIF18A KO cells form stable nuclear envelopes characterized by increased recruitment of nuclear envelope components and successful expansion of decondensing chromatin compared with those induced by nocodazole washout or radiation. Lagging chromosomes were also positioned closer to the main chromatin masses in KIF18A KO cells. These data suggest that not all micronuclei actively promote tumorigenesis.     

An electrophoretic capture method for efficient recovery of rare sequences from heterogeneous DNA

It is difficult to isolate rare, PCR-quality DNA from specimens containing large quantities of nonspecific DNA from multiple sources (heterogeneous DNA). Extracting human DNA from stool for colorectal cancer (CRC) screening tests exemplifies this technically challenging sample preparation problem. The stool matrix is complex, the DNA composition heterogeneous, and CRC-associated mutated DNA is rare. This report describes a novel solid phase DNA sequence-specific hybrid capture sample preparation method: the reversible electrophoretic capture affinity protocol (RECAP). We show that RECAP, compared with other methods, is capable of extracting linearly increasing amounts of human DNA from increasing amounts of total stool DNA in a manner that avoids co-purifying PCR inhibitors. RECAP thereby increases the yield of rare mutated DNA molecules and thus increases the detection sensitivity for CRC-associated mutations.     

原发性肝癌:CT血液供应、P53抗体和血管内皮生长因子

目的:分析原发性肝癌(primary hepatic carcinoma,简称肝癌)血液供应(blood supply,简称血供)CT类型与患者血清P53抗体、血管内皮生长因子(vascular endothelial growth factor,VEGF)的关系,以及血清P53抗体与VEGF的相关性.方法:用酶联免疫吸附法定量检测首诊肝癌患者血清P53抗体和VEGF,与CT诊断的血供类型结果进行对比,分析不同血供类型的肝癌是否存在血清P53抗体差异(单因素分析),并分析不同血供类型的肝癌血清VEGF是否存在差异(单因素分析),然后分析血清P53抗体和VEGF之间的相关性.结果:四种不同血供类型的肝癌之间血清P53抗体滴度有差异(P<0.05).值得注意的是,富血供型与混合型之间、动-静脉漏型(AVS)与混合型之间血清P53抗体有显著差异(P<0.05),富血供型与AVS型之间血清P53抗体没有显著差异(P>0.05).不同血供类型的肝癌中,富血供型和混合型血清P53抗体与VEGF呈正相关(P<0.05),而AVS型和乏血供型中,血清P53抗体与VEGF不呈直线相关(P>0.05).结论:血清P53抗体和血清VEGF滴度在血供不同肝癌中滴度有差异,在富血供型和混合型中血清P53抗体与VEGF滴度呈正相关.     

Analysis of Venezuelan equine encephalitis replicon particles packaged in different coats

Background

The Venezuelan equine encephalitis (VEE) virus replicon system was used to produce virus-like replicon particles (VRP) packaged with a number of different VEE-derived glycoprotein (GP) coats. The GP coat is believed to be responsible for the cellular tropism noted for VRP and it is possible that different VEE GP coats may have different affinities for cells. We examined VRP packaged in four different VEE GP coats for their ability to infect cells in vitro and to induce both humoral and cellular immune responses in vivo.

Methodology/Principal Findings

The VRP preparations were characterized to determine both infectious units (IU) and genome equivalents (GE) prior to in vivo analysis. VRP packaged with different VEE GP coats demonstrated widely varying GE/IU ratios based on Vero cell infectivity. BALB/c mice were immunized with the different VRP based on equal GE titers and the humoral and cellular responses to the expressed HIV gag gene measured. The magnitude of the immune responses measured in mice revealed small but significant differences between different GP coats when immunization was based on GE titers.

Conclusions/Significance

We suggest that care should be taken when alternative coat proteins are used to package vector-based systems as the titers determined by cell culture infection may not represent accurate particle numbers and in turn may not accurately represent actual in vivo dose.     

Lifestyle modifications to prevent and control hypertension. 1. Methods and an overview of the Canadian recommendations. Canadian Hypertension Society,Canadian Coalition for High Blood Pressure Prevention and Control,Laboratory Centre for Disease Control at Health Canada,Heart and Stroke Foundation of Canada

OBJECTIVE: To provide updated, evidence-based recommendations for health care professionals on lifestyle changes to prevent and control hypertension in otherwise healthy adults (except pregnant women). OPTIONS: For people at risk for hypertension, there are a number of lifestyle options that may avert the condition--maintaining a healthy body weight, moderating consumption of alcohol, exercising, reducing sodium intake, altering intake of calcium, magnesium and potassium, and reducing stress. Following these options will maintain or reduce the risk of hypertension. For people who already have hypertension, the options for controlling the condition are lifestyle modification, antihypertensive medications or a combination of these options; with no treatment, these people remain at risk for the complications of hypertension. OUTCOMES: The health outcomes considered were changes in blood pressure and in morbidity and mortality rates. Because of insufficient evidence, no economic outcomes were considered. EVIDENCE: A MEDLINE search was conducted for the period January 1996 to September 1996 for each of the interventions studied. Reference lists were scanned, experts were polled, and the personal files of the authors were used to identify other studies. All relevant articles were reviewed, classified according to study design and graded according to level of evidence. VALUES: A high value was placed on the avoidance of cardiovascular morbidity and premature death caused by untreated hypertension. BENEFITS, HARMS AND COSTS: Lifestyle modification by means of weight loss (or maintenance of healthy body weight), regular exercise and low alcohol consumption will reduce the blood pressure of appropriately selected normotensive and hypertensive people. Sodium restriction and stress management will reduce the blood pressure of appropriately selected hypertensive patients. The side effects of these therapies are few, and the indirect benefits are well known. There are certainly costs associated with lifestyle modification, but they were not measured in the studies reviewed. Supplementing the diet with potassium, calcium and magnesium has not been associated with a clinically important reduction in blood pressure in people consuming a healthy diet. RECOMMENDATIONS: (1) It is recommended that health care professionals determine the body mass index (weight in kilograms/[height in metres]2) and alcohol consumption of all adult patients and assess sodium consumption and stress levels in all hypertensive patients. (2) To reduce blood pressure in the population at large, it is recommended that Canadians attain and maintain a healthy body mass index. For those who choose to drink alcohol intake should be limited to 2 or fewer standard drinks per day (maximum of 14/week for men and 9/week for women). Adults should exercise regularly. (3) To reduce blood pressure in hypertensive patients, individualized therapy is recommended. This therapy should emphasize weight loss for overweight patients, abstinence from or moderation in alcohol intake, regular exercise, restriction of sodium intake and, in appropriate circumstances, individualized cognitive behaviour modification to reduce the negative effects of stress. VALIDATION: The recommendations were reviewed by all of the sponsoring organizations and by participants in a satellite symposium of the fourth international Conference on Preventive Cardiology. They are similar to those of the World Hypertension League and the Joint National committee, with the exception of the recommendations on stress management, which are based on new information. They have not been clinically tested. SPONSORS: The Canadian Hypertension Society, the Canadian Coalition for High Blood Pressure Prevention and Control, the Laboratory Centre for Disease Control at health Canada, and the Heart and Stroke Foundation of Canada.